DESCRIPTION (Adapted from applicants abstract): Listeria monocytogenes is a model facultative intracellular pathogen which has been widely used for the study of cell-mediated immunity. It is the cause of a serious disease especially in pregnant women, the newborn child, and immunocompromised adults. Recent studies in the laboratories of Drs. D. A. Portnoy and L. Tilney on the growth of this bacterium in tissue culture cells have defined the sequence of events involved with the intracellular growth and cell-to-cell spread of L. monocytogenes. These studies have provided a cell-biological explanation for the absolute requirement for cell-mediated immunity, i.e., the bacteria never leave the host cytoplasm, yet are able to spread cell-to-cell. A search for mutants defective for cell-to-cell spread resulted in the isolation of two which were localized in a previously identified open reading frame adjacent to the structural gene for L. monocytogenes hemolysin. Comparison of the amino acid sequence predicted by the newly defined gene, designated plcA, with sequences in a database revealed extensive homology with the Bacillus thuringiensis phosphatidylinositol-specific phospholipase C(PI-PLC). The investigator(s) have subsequently shown that L. monocytogenes culture fluids have an active PI-PLC, which is completely lacing in plcA mutants. These mutants are unable to propagate in host issues and have markedly decreased virulence in mice. This is the first demonstrated biological role for a bacterial PI-PLC. L. monocytogenes also secretes a broad specificity phospholipase C (PC-PLC). The goal of the proposed research is to purify the two phospholipases C and to characterize them with respect to their structures, amino acid sequences and their substrate specificities on lipid dispersions and with natural membranes. To precisely define the role of the PC-PLC in L. monocytogenes pathogenesis, mutants lacking this enzyme will be sought. Both PLCs will be characterized at the molecular genetic level. The role of the PLCs in the cell biology of infection will be investigated by studying: 1) effects of mutations in the genes on the pathogenesis of L. monocytogenes in mice; 2) effects of mutations on growth and cell-to-cell spread in tissue culture cells; and 3) the in vivo substrates for both PLCs. This research poses novel questions concerning the role of bacterial PLCs in a significant intracellular pathogen.